The overall goals of this research proposal are to determine the role(s) that individual amelogenin polypeptides play in enamel development, and how alterations in them may result in deranged amelogenesis, such as occurs in the heritable disease, Amelogenesis Imperfecta. With presently available techniques, solubilized amelogenin proteins can be resolved into 10 or more components. Although physiologic processing or artefactual degradation probably contribute to this heterogeneity, recent information from our laboratory suggests that at least some of the heterogeneity arises from the transcription of two divergent genes located on the X and Y chromosomes and from alternative splicing of the primary transcripts. To prove this, we have adopted a combined immunologic and protein analytic approach to isolate and characterize some of the individual components using high resolution chromatographic and electrophoretic techniques. Emphasis will be placed on identifying the translation products of alternatively spliced messages, and the resolved components will be subjected to extensive immunologic and chemical analyses to distinguish them from proteolytically processed components. the cloning and sequence analysis of the normal human amelogenin genes will be completed, and these sequences will be compared to those of genes, amplified by the polymerase chain reaction, in patients with Amelogenesis Imperfecta (AI) in order to better understand the defects involved at the molecular level. In addition, we plan to compare the functional characteristics of the normal and abnormal genes in transgenic mice. Finally, selected amelogenin polypeptides, including normal and mutated sequences that we design as well as peptides corresponding to those found in AI patients, will be expressed by recombinant DNA techniques, in order to evaluate functions of individual peptides, and to formulate and test hypotheses about the roles of amelogenins in enamel development.
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